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1 Nematode-Trapping in Pleurotus tuberregium D. S. Hibbett; R. G. Thorn Mycologia, Vol. 86, No. 5. (Sep. - Oct., 1994), pp Stable URL: Mycologia is currently published by Mycological Society of America. Your use of the JSTOR archive indicates your acceptance of JSTOR's Terms and Conditions of Use, available at JSTOR's Terms and Conditions of Use provides, in part, that unless you have obtained prior permission, you may not download an entire issue of a journal or multiple copies of articles, and you may use content in the JSTOR archive only for your personal, non-commercial use. Please contact the publisher regarding any further use of this work. Publisher contact information may be obtained at Each copy of any part of a JSTOR transmission must contain the same copyright notice that appears on the screen or printed page of such transmission. The JSTOR Archive is a trusted digital repository providing for long-term preservation and access to leading academic journals and scholarly literature from around the world. The Archive is supported by libraries, scholarly societies, publishers, and foundations. It is an initiative of JSTOR, a not-for-profit organization with a mission to help the scholarly community take advantage of advances in technology. For more information regarding JSTOR, please contact support@jstor.org. Tue Jan 8 10:01:

2 Mycologia, 86(5), 1994, pp O 1994, by The New York Botanical Garden, Bronx, NY Nematode-trapping in Pleurotus tuberregium D. S. Hibbett Harvard C'nzverszty Herbarza, 22 Drvznttj Avenue, Cambrzdge, Mmsachusetts R. G. Thorn Center for Mzcrobzal Ecology,,Vzchzgan State I'n~~~ersztj, East Lanszng, AMzchzgan Abstract: Pleurotus tuberregium is a common basidiomycete in parts of tropical Africa and Australia that fruits from a subterranean true sclerotium. Evidence from field collections suggests that the sclerotia are formed underground. Because its basidiocarps have intercalary skeletal hyphae, P. tuberregium has also been classified in both Lentinus and Panus. We investigated nematode-trapping in P. tuberregium in an attempt to resolve its generic placement. Aerial hyphae of P. tuberregium cultures on agar produced droplets of toxin on stalked secretory processes. Nematodes that came in contact with toxin droplets were paralyzed and then colonized by hyphae. This mode of nematode capture has been demonstrated previously only in Pleurotus sensu stricto, which supports classification of this species in Pleurotus. Key Words: Lentinus, n e m a t o p h a ~, Punus, Pleurotus tuberregium Most major groups of fungi are known to contain species that attack and consume nematodes by means of adhesive or ingested spores or various structures on vegetative hyphae. Hyphal modifications for nematode attack or capture include adheshe knobs, nets, stephanocysts, and filaments, constricting and nonconstricting rings, cells that secrete paralyzing toxin droplets, and mycelia that contain cytoplasmic toxins (Barron, 1977; Thorn and Barron, 1984; Barron and Thorn, 1987; Tzean and Liou, 1993). Clearly, nematode-trapping per se is a convergent character, but the particular modes and structures of capture may provide phylogenetically informative suites of characters. In this report we describe nematode-trapping ' Contribution No. 122 from the Christensen Research Institute, P.O. Box 305, Madang, Papua New Guinea. Accepted for publication May 13, in Pleurotus tuberregzum (Fr.) Singel- and discuss it5 taxonomic significance. - Pleurotus tuberregiunz occurs in Africa, India, and Australasia (Pegler, 1983). It is unusual among traditionally accepted Plez~rot~ls species (e.g., Hilber, 1982) in that its fruiting bodies arise not from wood, but from underground true sclerotia. Young mushrooms of P. tuberregium are eaten, but with age they develop a tough, leathery consistent)- and are no longer palatable (Corner, 1981). The toughness is due to the presence of numerous thick-walled skeletal hyphae that are especially well-developed in the stipe. Sclerotia of P. tuberregium are used as food, medicine, and as a source of additional crops of fruiting bodies (Zoberi, 1973; Oso, 1977; Corner, 1981; Pegler, 1983). The taxonomic history of P. tz~berregium reflects the lack of consensus regarding generic limits of Plezlrotu~ (Fr.) Kummer, Pa?zusFr., and Lentinus Fr. Singer (1951, 1986), whose concept of Plezlrotu~we follo~v, placed P. tuberregiunz in Pleurotus primarily on the basis of anatomy of the hymenophoral trama and subhymenium. However, Pegler (1975, 1983) restricted Pleurotus to monomitic species (those lacking skeletal or binding hyphae) and therefore excluded P. tuberregium, which he placed in Lentinus. Corner (1981) accepted some dimitic species in Pleurotu~,but only those \\.it11 strictly terminal skeletal hyphae. Pleurotus tuberr-egium, ~vhich Corner placed in Punus, has intercalar) and terminal skeletal hyphae. Information from studies of nucleic acids has helped refine the limits of Pleurotus, Lentinus, and Panus. The three genera are usually considered to be very closely related, but studies of ribosonlal RNA gene sequence data have suggested that the lentinoid-pleurotoid fungi actuallj- conlprise at least four independent lineages (Hibbett and Vilgalys, 1993). Cultural characters have also provided taxonomic resolution. Most lentinoid-pleurotoid fungi produce a white-rot, but there are also some brown-rot species in Lentinus s. lat. and Panus s. lat. (Gilbertson, 1981). The brown-rot species have been segregated as ~Veolentinus Redhead 8- Ginns and Heliocybe Redhead 8c Ginns (Redhead and Ginns, 1985). The species removed to LVeolentinusgenerally have weakly developed skeletal hyphae and are therefore anaton~ically intermediate between Lentinus s. str. and Punus s. str., which are strongly dimitic, and Pleurotz~s,which is mosth

3 monomitic. Thus, segregation of Neolentinus and Heliocybe, which was supported by the RNA sequence data (Hibbett and Vilgalys, 1993), helped to differentiate Pleurotus from both Lentinus and Panus (Redhead and Ginns, 1985). Members of Pleurotus sensu Singer (1975, 1986) display a method of nematode capture that appears to be unique to this genus (Thorn and Barron, 1984; Redhead and Ginns, 1985; Barron and Thorn, 1987; Petersen, 1993; Thorn and Tsuneda, 1993). In agar cultures, aerial hyphae of Pleurotus produce tiny droplets of nematode toxin on minute secretory processes. The toxin has been identified as trans-2-decenedioic acid (Kwok et al., 1992). Nematodes that come in contact with the toxin are paralyzed and later invaded by the Pleurotus hyphae. This mode of nematode attack has been observed in P. cornucopiae (Paulet) Rolland, P. cystidiosus O.K. Miller, P, levis (Berk. & Curt.) Sing., P. ostreatus Uacq. : Fr.) Kummer, P. populinus Hilber & Vilgalys (as P. ostreatus p.p. and P. subareolatus Peck), and P. pulmonarius (Fr. : Fr.) (2ui.l. (as P. ostreatus p.p.) (Thorn and Barron, 1984; Thorn and Tsuneda, 1993; redeterminations on the basis of mating studies, cf. L'ilgalys et al., 1993). In the course of screening isolates for nematode-trapping ability, several other lentinoidpleurotoid species have been tested for which the results are reported here for the first time. Pleurotus dryinus (Pers. : Fr.) Kummer (M. Tucic no. 2, TRTC), P. euosmus (Berk.) Sacc. (CBS ), and P. eryngii (D.C.: Fr.) Qui.1. (CBS ) demonstrated nematode-trapping ability typical of Pleurotus, but Lentinus suavissimus Fr. [DXOM , as Panus suavissimus (Fr.) Singer], L. tigrinus (Bull. : Fr.) Fr. (DAOM 3048), Panus lecomtei (Fr.) Corner (DAOM 21964, RGT /02, as P. rudis Fr.), Lentinula edodes (Berk.) Pegler (DAOM ) and Seolentinus lepideus (Fr.: Fr.) Redhead & Ginns (DAOM ) were all negative. The present study was intended to demonstrate whether Pleurotus tuberregium produces nematodetrapping toxin droplets, which we suggest may be a synapomorphy and diagnostic character for Pleurotus. The isolate of P. tuberregium used in this study (DSH ) was collected in October, 1992, in lowland rainforest at Amron, Madang Province, Papua New Guinea. The collection consists of a single basidiocarp that arises from a sclerotium 8 cm in diameter. The basidiocarp and sclerotium were separated and dried for approximately 12 h. In August, 1993, a portion of the sclerotium was excised and fragments were placed on MEA (1.25% malt extract, 2% agar) plates and incubated at room temperature. Clamped hyphae grew out from the sclerotial tissue within 2 days. MEA blocks with hyphal tips were transferred to W,4 (2% agar) plates and grown for 7-14 days, after which copious toxin droplet-pro- ducing secretory processes were visible (FIGS. 1-3). These structures have been called secretory cells (Barron and Thorn, 1987), but we have avoided this term because we could not see septa at their bases. Secretory processes were borne on aerial hyphae and usually were pointed more or less straight up, so that from the top only the round outlines of the toxin droplets were visible (FIG. 1).Viewed from the side, the secretory processes were composed of a slender stalk, leading to a rounded head about 1 Fm broad (FIGS. 2, 3). Live nematodes (Rhabditis sp., Carolina Biological Supply Co., catalog no. L268) were transferred directly to the mycelial mat or to the periphery of the mat. Nematodes swam easily over hyphae at the very periphery of the colony, where there were no toxin droplets. However, when nematodes struck toxin droplets on the older parts of the mat they usually recoiled immediately. Smaller nematodes were affected most profoundly; they quickly became sluggish, and the sinuous, energetic swimming movements were replaced by slow wagging of the head back and forth, ineffectual flexing of the entire body, and eventual paralysis, with only occasional weak movements of the body. As reported previously with P. ostreatus (Barron and Thorn, I&), the nematode's oesophagus became contorted and the shape of the head changed from elongate and gradually tapering to bluntly rounded after contact with toxin droplets (FIGS. 4, 5). Larger nematodes were not as susceptible to the toxin and some appeared to be completely unaffected. Nematodes that were paralyzed by the toxin droplets were colonized by hyphae after about The mode of nematode capture exhibited by P. tuberregium is essentially identical to that previously reported in other Pleurotus species (Thorn and Barron, 1984; Barron and Thorn, 1987), ~vhich suggests that this fungus is correctly classified in Pleurotus. There are also corroborating - anatomical features. Pleurotus tuberregium has large, smooth, cylindric, hyaline spores, that are typical of Pleurotus. Many Pleurotus species are monomitic, but skeletal hyphae are found in P. dryinus and P. levis. Perhaps the most unusual feature of P. tuberregium is the sclerotium from which fruiting bodies are produced. No other species presently classified in Plez~rotusproduces such a structure. However, sclerotia are produced by some species of Lentinus s. lat., including Lentinus dactyloides Clel., \\.hich is an Australian fungus that is dimitic with skeletal hyphae and that fruits from a subterranean, digitate sclerotium (Cleland, 1935; Pegler, 1983). Lentinus dactyloides has been shown to be closely related to Plezcrotus eryngii in cladistic analyses of ribosomal RNA sequence data (Hibbett and Vilgalys, 1993). Taken together, the molecular and morphological characters suggest that L. dactyloides may be properly classified in Pleurotus. Cul-

4 FIGS Nematode-trapping structures of Pleurotus tuberregium and nematodes on agar cultures of P. tuberregium. 1. Toxin droplets on aerial hyphae, viewed without coverslip. 2. Side view of toxin droplet on stalked appendage, viewed without coverslip. 3. Secretory process viewed in water mount under coverslip. The extracellular toxin droplet has been disrupted or dissolved. 4. Healthy, swimming nematode. 5. Nematode that has been paralyzed by contact with toxin droplet. Note that the head of the nematode has become bluntly rounded as compared to the head of the unaffected nematode in FIG. 4. Scale bars = 20 pm in FIG. 1; 10 pm in FIG. 2; 2 pm in FIG. 3; 50 pm in FIGS. 4 and 5. tures of L. dactyloides have not been tested for nematode-trapping ability. Both Corner (1981) and Pegler (1983) have suggested that P. tuberregium is part of a group of closely related species with an Australasian-African distribution. Species that are putatively closely related to P. tuberregium include Lentinus connutus Berk., L. anthocephulus (Liv.) Pegler, and L. fusipes Cooke & Massee, all of which are in Lentinus sect. Tuberregium (Singer) Pegler (Pegler, 1983). Another species of Lentinus s. lat. that may in fact be a Pleurotus is L. giganteus Berk. which occurs in tropical Asia and Australia (Comer, 1981; Pegler, 1983). Lentinus giganteus fruits on the ground and has a deeply radicating stipe that originates from buried wood (Corner, 1981; Pegler, 1983). Similarities of L. giganteus to Pleurotus, including marcescent fruiting bodies and the presence of capitate, lecythiform cystidia, were discussed by Comer (1981) and Pegler (1983). Future studies aimed at understanding the monophyletic limits of Pleurotus should consider these species as well as L. dactyloides, and should test their cultures for nematode-trapping ability. Our final note concerns formation of the sclerotium in P. tuberregium. Comer (1981) and Pegler (1983) stated that the sclerotia of P. tuberregium develop above ground in logs, then fall to the ground and become covered with soil. Our observations of P. tuberregium, which was abundant at Amron, do not support this view. The sclerotium that we collected was buried approximately 10 cm underground. All of the fruiting bodies that we saw grew out of densely packed soil with a thin litter layer and no evidence of disturbance. It is most probable that the sclerotia were formed underground. Field work in Papua New Guinea was supported in part by a Papua New Guinea University of Technology (UNITECH) Visiting Research Fellowship and a Science and Technology Agency of Japan Postdoctoral Fellowship to DSH, and by assistance from the Christensen Research Institute (CRI). DSH especially thanks Chris Mercer of UNITECH and Matthew Jebb of CRI for assistance in the field, logistical support, and access to herbarium facilities. We also thank Donald Pfister and Michael Donoghue (Harvard University Herbaria) for providing facilities to DSH for microscopy and culturing, George L. Barron (University of Guelph, Canada) for permission to report unpublished results of RGT

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